Abstract:

Objective  To construct the prokaryotic expression plasmid of human high mobility group box1 (hHMGB1) gene,  and to express and purify the His-tagged hHMGB1 fusion protein. Methods  According to the sequence reported in GenBank, we optimized codons by OptimumGeneTM and synthesized the human HMGB1 gene. To construct prokaryotic expression plasmid pQE-T7-2/HMGB1, the DNA fragment obtained from PCR was digested with NdeⅠ and XhoⅠ, and then inserted into pQE-T7-2 that was cut with the same enzymes. The recombinants were screened by colony PCR, restriction mapping and sequencing. The recombinant E.coli BL21 (DE3) was induced for expression with isopropyl-beta-D-thiogalactopyranoside (IPTG), and the recombinant protein was identified by SDS-PAGE and Western blotting. The recombinant human HMGB1 was further purified by Ni-NTA Agarose. Results  The prokaryotic expression plasmid pQE-T7-2/HMGB1 was successfully constructed. The expressed recombinant protein accounted for about 20% of the total bacterial proteins. The recombinant protein could specifically react with antiHMGB1 antibody and anti-His antibody. The purity of recombinant human HMGB1 separated with affinity chromatography was more than 90%. Conclusion  The recombinant prokaryotic expression vector of human HMGB1 which was successfully constructed may lay the foundation for the  further study of the function of human HMGB1.

Key words: High mobility group proteins； Escherichia coli； Prokaryotic cells； Purification